My long-term goal is to understand the molecular biology and regulation of inositol 1,4,5-trisphosphate receptors (IP3Rs) and, in particular, their processing by the ER-associated degradation (ERAD) pathway, a facet of the ubiquitin-proteasome pathway. In recent years we have discovered (i) that a very large (~2MDa) complex composed of two ER membrane proteins, termed erlin1 and erlin2, binds directly to activated IP3Rs and mediates their ERAD, (ii) that the erlin1/2 complex binds to phosphatidylinositol 3-phosphate (PI(3)P), a lipid critical to intracellular organelle development and trafficking, and (iii) that human neurodegenerative disease-linked point mutations to erlin2 alter the functionality of the complex. The key unresolved questions that flow from these findings are as follows: how does the erlin1/2 complex recognize activated IP3Rs and target them for ERAD, what is the full significance of PI(3)P binding to the erlin1/2 complex, and how do naturally-occurring point mutations to the erlin1/2 complex alter its properties? These questions will be addressed through 2 Specific Aims. Aim 1) Analysis of how the erlin1/2 complex and activated IP3Rs interact The approach taken will be to express (i) erlin2, or (ii) IP3R1 mutants in cells lacking these proteins and assess the extent to which the mutations impair the interaction between activated IP3R1 and the erlin1/2 complex, (iii) identify the interacting regions by cross-linking followed by mass spectrometry, and (iv) generate a high resolution structural model of the erlin1/2 complex using cryo-EM and NMR spectroscopy that can be docked with an equivalent structure of activated IP3R1. Results obtained from these complementary approaches should identify the interaction points between activated IP3R1 and the erlin1/2 complex and establish what it is about IP3R1 tetramer activation that triggers its recognition by the erlin1/2 complex for ERAD. This will illuminate a key step in the life cycle of IP3Rs and will provide the first detailed description of how a native endogenous signaling protein is recognized for processing by the ERAD pathway. Aim 2) Analysis of the PI(3)P-binding capacity of the erlin1/2 complex and its significance The approach taken will be to determine (i) how PI(3)P binds to the erlin1/2 complex from mutagenesis and NMR spectroscopy, (ii) whether PI(3)P binding to the erlin1/2 complex is required for IP3R ERAD, and (iii) how the erlin1/2 complex affects autophagosome formation, or other PI(3)P-dependent cellular processes. Results obtained will illuminate a completely novel aspect of erlin1/2 complex biology and should identify new mechanistic details about IP3R ERAD and PI(3)P-dependent cellular processes. In summary, novel topics will be investigated for the first time, with outcomes that will advance our understanding of the IP3R-erlin1/2 complex-RNF170 axis, ERAD in general and neurodegenerative disorders.